The ability of myosin to generate force and motion through its interaction with actin filaments is essential to many biological processes including muscle contraction, cell division, and intracellular transport. The atomic level structures of myosin in various stages of its enzymatic cycle have provided a framework of the molecular mechanism of force generation utilized by myosin. These structures as well as other biochemical and structural data suggest that myosin generates force by coupling small conformational changes in the nucleotide-binding region to a large swing of the light-chain binding region while myosin is strongly bound to actin. However, there is a lack of information about the structural details of how myosin alters its affinity for actin throughout its ATPase cycle, and how actin-binding activates the dissociation of the products of ATP hydrolysis (ADP and phosphate), which triggers force production. The current proposal hypothesizes that the large cleft that separates the actin-binding domain changes conformation rapidly to allow binding to actin prior to phosphate release and force generation. Moreover, the switch II region in the nucleotide-binding domain is hypothesized to directly couple conformational changes to the lever arm. Myosin V, a non-muscle myosin that has unique structural and biochemical properties, will be used as a model to examine specific conformational changes in the actin- and nucleotide-binding regions of myosin. Intrinsic and extrinsic fluorescence probes will be strategically placed to measure conformational changes in the actin-, nucleotide-binding, and lever arm regions during the enzymatic cycle of myosin. In addition, transient kinetic experiments will be used to correlate the conformational changes with specific biochemical steps in the actomyosin ATPase cycle. We will use computational methods to propose a conformational pathway of the myosin ATPase cycle consistent with our experimental data. By integrating the computational and experimental data we will elucidate critical details about the structural mechanism of force generation in myosin and further our understanding of genetic diseases associated with point mutations in myosin, such as Familial Hypertrophic Cardiomyopathy. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine how myosin converts chemical energy into force and motion to drive the process of muscle contraction. A combination of experimental and computational biophysical tools will be utilized to define the structural pathway of the actomyosin V ATPase cycle, which will fill in critical gaps in what is known about how myosin generates force in muscle contraction. Since point mutations in myosin are associated with genetic diseases such as Familial Hypertrophic Cardiomyopathy, elucidating the structural pathway for energy transduction in myosin may improve our understanding of and lead to future treatments for these diseases.